Abstract
Studies were carried out on microsomes of the digestive gland of the common mussel, Mytilus edulis L. Cytochrome P-450 specific content, and the specific contents or activities of other mixed-function oxidase (MFO) components, and the oxidative activities benzo[a]pyrene hydroxylase (BPH) and NADPH-independent 7-ethoxycoumarin O-deethylase (ECOD), all varied seasonally. To varying extents, correlations were seen between changes in these parameters, and changes in the mussel reproductive cycle and/or the seasonal variation in water temperature. The existence of P-450 isoenzymes was indicated by asynchrony in the seasonal changes in BPH and NADPH-independent ECOD activities relative to the changes in P-450 specific content, and by seasonal changes in cytochrome P-450 A max and the microsomal protein profile on SDS PAGE. Indications of P-450 isoenzymes were also obtained from purification studies, and from kinetic studies of NADPH-independent ECOD activity (multiphasic kinetics seen with 7-ethoxycoumarin concentration). The purification scheme essentially comprised sodium cholate solubilization, (NH4)2SO4-protein fractionation and affinity and ion-exchange column chromatographic steps. Two cytochrome P-450 peaks were obtained on DEAE-sephacel ion-exchange chromatography (KC1 elution), the overall purification for the major peak being x 20, with a yield of 5%. The final detergent-free P-450 preparation was largely in the low-spin state, and had a monomeric molecular weight of 53.0 Kd. Ligand-binding experiments were performed on partially-purified cytochrome P-450 preparations. Type II spectra were obtained with N-substituted imidazole compounds, metyrapone and pyridine, but with compounds giving type I spectra with mammalian cytochromes P-450 (testosterone, 7-ethoxycoumarin, a-naphthoflavone and SKF 525-A), reverse type I spectra were seen. In vitro MFO metabolic activity toward possible xenobiotic and endogenous substrates, was limited, and surprisingly, largely NADPH-independent. NADPH-independent ECOD activity was susceptable to modulators of mammalian MFO activity, and indicated to be cytochrome P-450-mediated. Mussel microsomal fraction and microsomal-extract were shown to inhibit rat MFO activities and hexobarbital binding to rat cytochrome P-450. These and other results are discussed in terms of a possible "endogenous blocking" of the substrate-binding site of the mussel P-450, and in terms of possible mechanisms of cytochrome P-450 catalytic action in addition to monooxygenation, such as peroxidation. Mussel cytochrome P-450 specific content was relatively unaffected by a variety of mammalian model P-450 inducers, with the exception of a small elevation with exposure to 3-methylcholanthrene (3MC). In contrast, NADPH-cytochrome c (P-450) reductase activity was slightly more responsive. An increase in NADPH-independent ECOD activity with 3MC-exposure was seen at one time of the year, but not at other times.